quiz 4 MOD 5

Question 1

What is a barcode? What are barcodes used for?

Answer 1

A DNA barcode is a short, standardized DNA sequence used to identify species. It is analogous to the barcode used for product identification.
Barcodes are used in various fields like ecology, forensic science, and species identification to quickly and accurately identify species.

Question 2

List the characteristics of a good barcode sequence

Answer 2

Short: Typically 500-800 base pairs.
Variable: Contains regions that vary between species but remain consistent within species.
Easy to amplify: PCR amplification should be straightforward.
Unique: No two species should have identical barcode sequences.
Conserved flanking regions: Allows for universal primer design.

Question 3

Both morphological characters and DNA sequences are used to build trees. Which is better?

Answer 3

DNA sequences are generally considered more reliable due to their direct measure of genetic similarity, while morphological characters can be influenced by environmental factors.

Question 4

List the different types of data that can be used to build a phylogenetic tree

Answer 4

Morphological data: Physical characteristics.
Molecular data: DNA, RNA sequences.
Biochemical data: Protein sequences, metabolic pathways.
Behavioral data: Mating rituals, social structures.
Fossil records: Morphological data from extinct species.

Question 5

Contrast between phylogeny and taxonomy

Answer 5

Phylogeny is the study of evolutionary relationships among species or other groups. It shows the branching pattern of evolution.
Taxonomy is the classification of organisms into hierarchical categories based on shared characteristics. It provides a system for naming and organizing species.

Question 6

Procedure/Difference from cheek cell extraction Silica based animal DNA Extraction

Answer 6

Procedure: Grinding tissue, lysing with GuHCl, heating, binding DNA to silica resin, centrifugation, washing with ethanol.
Difference from cheek cell extraction: Similar in principle but uses specific reagents and procedures optimized for animal tissue.

Question 7

Polymerase Chain Reaction (PCR) and Cleanup
gDNA (genomic DNA) vs mtDNA (mitochondrial DNA) - which is easier for PCR. Why do we use a mitochondrial or ribosomal genes for barcoding?
Genes used and why do we use this gene? Function of the protein coded
by this gene?
Multiplex PCR - Why do we use primer cocktails?

Answer 7

gDNA vs mtDNA: mtDNA is easier to amplify as it has higher copy numbers and is less fragmented.
Genes used: COI (Cytochrome c Oxidase subunit I), involved in electron transport in mitochondria.
Multiplex PCR: Uses multiple primer sets in one reaction to amplify different target regions simultaneously.

Question 8

PCR Cleanup or Purification (ExoSAP-IT)
○ Why is it necessary to clean up a PCR?
○ How does EXOSAP (function of the actual enzymes) clean up the PCR?
○ What is the optimal temperature for these enzymes?
○ Why is it important to denature the enzyme at 80oC before proceeding to
sequencing?

Answer 8

Necessity of PCR cleanup:
To remove unincorporated primers and nucleotides that can interfere with sequencing.
EXOSAP-IT enzymes:
Exonuclease I removes primers, and Shrimp Alkaline Phosphatase dephosphorylates nucleotides.
Optimal temperature:
37°C for initial digestion and 80°C for enzyme denaturation.C for enzyme denaturation.
important to inactivate the enzyme and prevent it from interfering with the sequencing reaction.

Question 9

DNA Sequencing and Sequence analysis - Chain Termination method
○ Understand the process and components of Sanger sequencing reaction
○ Compare PCR in Sanger sequencing and normal PCR
○ Polyacrylamide vs agarose gel
○ Reading a sequencing gel and the electropherogram to determine the
sequence of a template
○ Be able to determine sequence quality (Phred scores)

Answer 9

Sanger sequencing:
Uses dideoxynucleotides to terminate DNA synthesis at specific points.
PCR vs. Sanger sequencing:
Sanger sequencing uses dideoxynucleotides to stop elongation randomly.
Gel type:
Polyacrylamide for high-resolution, agarose for larger fragments.
Reading a sequencing gel:
Bands represent DNA fragments of varying lengths.
Sequence quality (Phred scores):
Indicates the probability of a base being called incorrectly.

Question 10

Phylogenetic Tree construction (DNA subway activity)
○ Sequence viewer in DNA subway ( purpose of sequence trimming excess
Ns)
○ Function of BLAST - Definition (e-values, Bit scores, query vs hits,
importance of # mismatches)
○ Function of MUSCLE - consensus sequence, sequence similarities (%),
trimming unaligned ends. How is this used to build a tree?
○ Function of PHYLIP (NJ or ML) - a gene tree (node, branches, clade,
taxa, outgroup) –
○ Be able to interpret a phylogenetic tree
○ Choosing an outgroup for tree building

Answer 10

Sequence viewer:
Trims low-quality regions and excess Ns from sequences.
BLAST:
Compares sequences to known databases to find similarities.
MUSCLE:
Aligns sequences to identify similarities and differences.
PHYLIP:
Software for tree construction using Neighbor Joining (NJ) or Maximum Likelihood (ML) methods.
Interpreting a tree:
Nodes represent common ancestors, branches indicate divergence, clades are groups of related taxa.
Choosing an outgroup:
A closely related species or group used to root the tree.